The term cancer applies to a group of diseases in which cells grow abnormally and form a malignant tumor. Malignant cells can invade nearby tissues and metastasize (i.e., travel to other sites in the body, where they establish secondary areas of growth). This aberrant growth pattern results from mutations in genes that regulate proliferation, differentiation, and survival of cells in a multicellular organism. Because of these genetic changes, cancer cells no longer respond to the signals that govern growth of normal cells (Fig. 17.1.)

Oncogenes and Tumor-Suppressor Genes. The genes involved in the development of cancer are classified as oncogenes or tumor-suppressor genes. Oncogenes are mutated derivatives of normal genes (proto-oncogenes), which function to promote proliferation or cell survival. These genes can code for growth factors, growth-factor receptors, signal transduction proteins, intracellular kinases, and transcription factors. The process of transformation into a malignant cell may begin with a gain-of-function mutation in only one copy of a proto-oncogene. As the mutated cell proliferates, additional mutations can occur. Tumor-suppressor genes (normal growth–suppressor genes) encode proteins that inhibit proliferation, promote cell death, or repair DNA; both alleles need to be inactivated for transformation (a loss of function). Growth-suppressor genes have been called the guardians of the cell.

Cell-Cycle Suppression and Apoptosis. Normal cell growth depends on a balanced regulation of cell-cycle progression and apoptosis (programmed cell death) by proto-oncogenes and growth-suppressor genes. At checkpoints in the cell cycle, products of tumor-suppressor genes slow growth in response to signals from the cell’s environment, including external growth-inhibitory factors, or to allow time for repair of damaged DNA, or in response to other adverse circumstances in cells. Alternatively, cells with damaged DNA are targeted for apoptosis so that they will not proliferate. Many growth-stimulatory pathways involving proto-oncogenes, and growth-inhibitory controls involving a variety of tumor-suppressor genes, converge to regulate the activity of some key protein kinases, the cyclin-dependent kinases (CDKs). These kinases act to control progression at specific points in the cell growth cycle. Apoptosis is initiated by either death-receptor activation or intracellular signals leading to release of the mitochondrial protein, cytochrome c.

Mutations. Mutations in DNA that give rise to cancer may be inherited or may be caused by chemical carcinogens, radiation, viruses, and by replication errors that are not repaired. A cell population must accumulate multiple mutations for transformation to malignancy.

Treatment. Cancer has been treated through a variety of approaches, including surgery, chemotherapy, and most recently molecular analysis of an individual’s cancer cells such that a personalized treatment plan can be developed. Molecular techniques (as described in the previous chapter) are more frequently being used to attack cancer at the molecular level.

THE WAITING ROOM

Mannie W. has chronic myelogenous leukemia (CML), a disease in which a single line of myeloid cells in the bone marrow proliferates abnormally, causing a large increase in the number of nonlymphoid white blood cells (see Chapter 15). His myeloid cells contain the abnormal Philadelphia chromosome, which increases their proliferation. He has recently complained of pain and tenderness in various areas of his skeleton, possibly stemming from the expanding mass of myeloid cells within his bone marrow. He also reports a variety of hemorrhagic signs, including bruises (ecchymoses), bleeding gums, and the appearance of small red spots (petechiae caused by release of red cells into the skin).

Michael T. was diagnosed with a poorly differentiated adenocarcinoma of the lung (see Chapter 12) after resection of a nodule very concerning for malignancy seen on a computed tomography (CT) scan of his chest. He survived the surgery and was recovering uneventfully until 6 months later, when he complained of an increasingly severe right temporal headache. A CT scan of his brain was performed. Results indicated that the cancer, which had originated in his lungs, had metastasized to his brain.

Clark T. has had an intestinal adenocarcinoma resected, as well as several small metastatic nodules in his liver (see Chapter 11). He completed his second course of chemotherapy with 5-fluorouracil (5-FU) and oxaliplatin and had no serious side effects. He assured his physician at his most recent checkup that, this time, he intended to comply with any instructions his physicians gave him. He ruefully commented that he wished he had returned for regular examinations after his first colonoscopy.

Calvin A. returned to his physician after observing a brownish-black irregular mole on his forearm (see Chapter 12). His physician thought the mole looked suspiciously like a malignant melanoma, so referred him to a dermatologist, who performed an excision biopsy (surgical removal for cytological analysis).

I. Causes of Cancer

The term cancer applies to a group of diseases in which cells grow abnormally and form a malignant tumor. Malignant cells can invade nearby tissues and metastasize (i.e., travel to other sites in the body where they establish secondary areas of growth). This aberrant growth pattern results from mutations in genes that regulate proliferation, differentiation, and survival of cells in a multicellular organism. Because of these genetic changes, cancer cells no longer respond to the signals that govern growth of normal cells.

Normal cells in the body respond to signals, such as cell–cell contact (contact inhibition), that direct them to stop proliferating. Cancer cells do not require growth-stimulatory signals, and they are resistant to growth-inhibitory signals. They are also resistant to apoptosis, the programmed cell death process whereby unwanted or irreparably damaged cells self-destruct. They have an infinite proliferative capacity and do not become senescent (i.e., they are immortalized). Furthermore, they can grow independent of structural support, such as the extracellular matrix (loss of anchorage dependence).

The study of cells in culture was, and continues to be, a great impetus for the study of cancer. Tumor development in animals can take months, and it was difficult to do experiments with tumor growth in animals. Once cells could be removed from an animal and propagated in a tissue culture dish, the onset of transformation (the normal cell becoming a cancer cell) could be seen in days.

Once cells were available to study, it was important to determine the criteria that distinguish transformed cells from normal cells in culture. Three criteria were established. The first is the requirement for serum in the cell culture medium to stimulate cell growth. Serum is the liquid fraction of clotted blood and contains many factors that stimulate cell proliferation. Transformed cells have, in general, a reduced requirement for serum: approximately 10% that is required for normal cells to grow. The second criterion is the ability to grow without attachment to a supporting matrix (anchorage dependence). Normal cells (such as fibroblasts or smooth muscle cells) require adherence to a substratum (in this case, the bottom of the plastic dish) and will not grow if suspended in a soft agar mixture. Transformed cells, however, have lost this anchorage dependence. The third and most stringent criterion used to demonstrate that cells are truly transformed is the ability of cells to form tumors when they are injected into mice that lack an immune system. Transformed cells will do so, whereas normal cells will not.

The transformation of a normal cell to a cancer cell begins with damage to DNA (base changes or strand breaks) caused by chemical carcinogens, UV light, viruses, or replication errors (see Chapter 12). Mutations result from the damaged DNA if it is not repaired properly or if it is not repaired before replication occurs. A mutation that can lead to transformation also may be inherited. When a cell with one mutation proliferates, this clonal expansion (proliferation of cells arising from a single cell) results in a substantial population of cells containing this one mutation, from which one cell may acquire a second mutation relevant to control of cell growth or death. With each clonal expansion, the probability of another transforming mutation increases. As mutations accumulate in genes that control proliferation, subsequent mutations occur even more rapidly, until the cells acquire the multiple mutations (in the range of four to seven) necessary for full transformation.

The transforming mutations occur in genes that regulate cellular proliferation and differentiation (proto-oncogenes), suppress growth (tumor-suppressor genes), target irreparably damaged cells for apoptosis, or repair damaged DNA. The genes that regulate cellular growth are called proto-oncogenes, and their mutated forms are called oncogenes. The term oncogene is derived from the Greek word “onkos,” meaning bulk or tumor. A transforming mutation in a proto-oncogene increases the activity or amount of the gene product (a gain-of-function mutation). Tumor-suppressor genes (normal growth-suppressor genes) and repair enzymes protect against uncontrolled cell proliferation. A transforming mutation in these protective genes results in a loss of activity or a decreased amount of the gene product.

In summary, cancer is caused by the accumulation of mutations in the genes involved in normal cellular growth and differentiation. These mutations give rise to cancer cells that are capable of unregulated, autonomous, and infinite proliferation. As these cancer cells proliferate, they impinge upon normal cellular functions, leading to the symptoms exhibited by individuals with the tumors.

II. Damage to DNA Leading to Mutations

An alteration in the chemical structure of DNA, or of the sequence of bases in a gene, is an absolute requirement for the development of cancer. The function of DNA depends on the presence of various polar chemical groups in DNA bases, which are capable of forming hydrogen bonds between DNA strands or other chemical reactions. The oxygen and nitrogen atoms in DNA bases are targets for a variety of electrophiles (electron-seeking chemical groups). A typical sequence of events leading to a mutation is shown for dimethylnitrosamine in Figure 17.2. Chemical carcinogens (compounds that can cause transforming mutations) found in the environment and ingested in foods are generally stable lipophilic compounds that, like dimethylnitrosamine, must be activated by metabolism in the body to react with DNA (see also benz[o]pyrene, Action of Mutagens, Chapter 12, Section III.A, and Fig. 12.12). Many chemotherapeutic agents, which are designed to kill proliferating cells by interacting with DNA, may also act as carcinogens and cause new mutations and tumors while eradicating the old. Structural alterations in DNA also occur through radiation and through UV light, which causes the formation of pyrimidine dimers. More than 90% of skin cancers occur in sunlight-exposed areas. UV rays derived from the sun induce an increased incidence of all skin cancers, including squamous cell carcinoma, basal cell carcinoma, and malignant melanoma of the skin. The wavelength of UV light that is most associated with skin cancer is UVB (280 to 320 nm), which forms pyrimidine dimers in DNA. This type of DNA damage is repaired by nucleotide excision repair pathways that require products of at least 20 genes. With excessive exposure to the sun, the nucleotide excision repair pathway is overwhelmed, and some damage remains unrepaired.

Each chemical carcinogen or reactant creates a characteristic modification in a DNA base. The DNA damage, if not repaired, introduces a mutation into the next generation when the cell proliferates.

• Radiation and chemical carcinogens act (1) by causing a mutation in the regulatory region of a gene, increasing the rate of production of the proto-oncogene protein, or (2) by producing a mutation in the coding portion of the oncogene that results in the synthesis of a protein of slightly different amino acid composition capable of transforming the cell (Fig. 17.3A).

  
  

  

  
  
• The entire proto-oncogene or a portion of it may be transposed or translocated, that is, moved from one position in the genome to another (see Fig. 17.3B). In its new location, the proto-oncogene may be under the control of a promoter that is regulated differently than the promoter that normally regulates this gene. This may allow the gene to be expressed in a tissue where it is not normally expressed or at higher-than-normal levels of expression. If only a portion of the proto-oncogene is translocated, it may be expressed as a truncated protein with altered properties, or it may fuse with another gene and produce a fusion protein containing portions of what are normally two separate proteins. The truncated or fusion protein may be hyperactive and cause inappropriate cell growth.
  
• The proto-oncogene may be amplified (see Fig. 17.3C), so that multiple copies of the gene are produced in a single cell. If more genes are active, more proto-oncogene proteins will be produced, increasing the growth rate of the cells. As examples, the oncogene N-myc (a cell proliferation transcription factor, related to c-myc) is amplified in some neuroblastomas, and amplification of the erb-B2 oncogene (a growth-factor receptor) is associated with several breast carcinomas.
  
• If an oncogenic virus infects a cell, its oncogene may integrate into the host-cell genome, permitting production of the abnormal oncogene protein. The cell may be transformed and exhibit an abnormal pattern of growth. Rather than inserting an oncogene, a virus may simply insert a strong promoter into the host-cell genome. This promoter may cause increased or untimely expression of a normal proto-oncogene.

The important point to remember is that transformation results from abnormalities in the normal growth-regulatory program caused by gain-of-function mutations in proto-oncogenes. However, loss-of-function mutations also occur in the tumor-suppressor genes, repair enzymes, or activators of apoptosis, and a combination of both types of mutations is usually required for full transformation to a cancer cell.

C. Mutations in Repair Enzymes

Repair enzymes are the first line of defense preventing conversion of chemical damage in DNA to a mutation (see Chapter 12, Section III.B). DNA repair enzymes are tumor-suppressor genes in the sense that errors repaired before replication do not become mutagenic. DNA damage is constantly occurring from exposure to sunlight, background radiation, toxins, and replication errors. If DNA repair enzymes are absent, mutations accumulate much more rapidly, and once a mutation develops in a growth-regulatory gene, a cancer may arise. As an example, inherited mutations in the tumor-suppressor genes BRCA1 and BRCA2 predispose women to the development of breast cancer (see Biochemical Comments at the end of this chapter). The protein products of these genes play roles in DNA repair, recombination, and regulation of transcription. A second example, HNPCC (hereditary nonpolyposis colorectal cancer), was introduced in Chapter 12. It results from inherited mutations in enzymes involved in the DNA mismatch repair system.

III. Oncogenes

Proto-oncogenes control normal cell growth and division. These genes encode proteins that are growth factors, growth-factor receptors, signal transduction proteins, transcription factors, cell-cycle regulators, and regulators of apoptosis (Table 17.1). (The name representing the gene of an oncogene is referred to in lowercase letters and italics [e.g., myc], but the name of the protein product is capitalized and italics are not used [e.g., Myc].) The mutations in oncogenes that give rise to transformation are usually gain-of-function mutations; either a more active protein is produced or an increased amount of the normal protein is synthesized.

TABLE 17.1 Classes of Oncogenes, Mechanism of Activation, and Associated Human Tumors

MicroRNAs (miRNAs) can also behave as oncogenes. If a miRNA is overexpressed (increased function), it can act as an oncogene if its target (which would exhibit reduced expression under these conditions) is a protein, which is involved in inhibiting, or antagonizing, cell proliferation.

A. Oncogenes and Signal Transduction Cascades

All of the proteins in growth-factor signal transduction cascades are coded for by proto-oncogenes (Fig. 17.4).

1. Growth Factors and Growth-Factor Receptors

Growth factors generally regulate growth by serving as ligands that bind to cellular receptors located on the plasma membrane (cell-surface receptors) (see Chapter 10). Binding of ligands to these receptors stimulates a signal transduction pathway in the cell that activates the transcription of certain genes. If too much of a growth factor or a growth-factor receptor is produced, the target cells may respond by proliferating inappropriately. Growth-factor receptors may also become oncogenic through translocation or point mutations in domains that affect binding of the growth factor, dimerization, kinase activity, or some other aspect of their signal transmission. In such cases, the receptor transmits a proliferative signal even though the growth factor normally required to activate the receptor is absent. In other words, the receptor is stuck in the “on” position.

2. Signal Transduction Proteins

The genes that encode proteins involved in growth-factor signal transduction cascades may also be proto-oncogenes. Consider, for example, the monomeric G-protein Ras. Binding of growth factor leads to the activation of Ras (see Fig. 10.17). When Ras binds guanosine triphosphate (GTP), it is active, but Ras slowly inactivates itself by hydrolyzing its bound GTP to guanosine diphosphate (GDP) and inorganic phosphate (P_i). This controls the length of time that Ras is active. Ras is converted to an oncogenic form by point mutations that decrease the activity of the GTPase domain of Ras, thereby increasing the length of time it remains in the active form.

Ras, when it is active, activates the serine–threonine kinase Raf (a mitogen-activated protein [MAP] kinase kinase kinase), which activates MEK (a MAP kinase kinase), which activates MAP kinase (Fig. 17.5). Activation of MAP kinase results in the phosphorylation of cytoplasmic and nuclear proteins, followed by increased transcription of the transcription-factor proto-oncogenes myc and fos (see below). Note that mutations in the genes for any of the proteins that regulate MAP kinase activity, as well as those proteins induced by MAP kinase activation, can lead to uncontrolled cell proliferation.

Many transcription factors, such as Myc and Fos, are proto-oncoproteins (the products of proto-oncogenes). MAP kinase, in addition to inducing myc and fos, also directly activates the AP-1 transcription factor through phosphorylation (see Fig. 17.5). AP-1 is a heterodimer formed by the protein products of the fos and jun families of proto-oncogenes. The targets of AP-1 activation are genes involved in cellular proliferation and progression through the cell cycle, as are the targets of the myc transcription factor. The synthesis of the transcription factor c-myc is tightly regulated in normal cells, and it is expressed only during the S phase of the cell cycle. In a large number of tumor types, this regulated expression is lost, and c-myc becomes inappropriately expressed or overexpressed throughout the cell cycle, driving cells continuously to proliferate.

The net result of alterations in the expression of transcription factors is the increased production of the proteins that carry out the processes required for proliferation.

B. Oncogenes and the Cell Cycle

Growth factors, hormones, and other messengers activate the growth of human cells, involving DNA replication and cell division in the cell cycle. These activators work through cyclins and CDKs that control progression from one phase of the cycle to another (Fig. 17.6). For quiescent cells to proliferate, they must leave G₀ and enter the G₁ phase of the cell cycle (see Chapter 12, Fig. 12.7). If the proper sequence of events occurs during G₁, the cells enter the S phase and are committed to DNA replication and cell division. Similarly, during G₂, cells make a commitment to mitotic division. CDKs are made continuously throughout the cell cycle but require binding of a specific cyclin to be active. Different cyclins made at different times in the cell-cycle control each of the transitions (G₁/S, S/G₂, G₂/M).

The activity of the cyclin–CDK complex is further regulated through phosphorylation and through inhibitory proteins called cyclin-dependent kinase inhibitors (CKIs) (Fig. 17.7). CKIs slow cell-cycle progression by binding and inhibiting the CDK–cyclin complexes. CDKs are also controlled through activating phosphorylation by CAK (cyclin-activating kinases) and inhibitory hyperphosphorylation kinases.

To illustrate the role of these proteins, consider some of the events that occur at the G₁/S checkpoint (Fig. 17.8). Because the cell is committed to DNA replication and division once it enters the S phase, multiple regulatory proteins are involved in determining whether the cell is ready to pass this checkpoint. These regulatory proteins include cdk4 and cdk6 (which are constitutively produced throughout the cell cycle), cyclin D (whose synthesis is induced only after growth-factor stimulation of a quiescent cell), the retinoblastoma gene product (Rb), and a class of transcription factors known collectively as E2F. In quiescent cells, Rb is complexed with E2F, resulting in inhibition of these transcription factors. Upon growth-factor stimulation, the cyclin Ds are induced (there are three types of cyclin D: D1, D2, and D3). They bind to cdk4 and cdk6, converting them to active protein kinases. One of the targets of cyclin/cdk phosphorylation is the Rb protein. Phosphorylation of Rb releases it from E2F, and E2F is then free to activate the transcription of genes required for entry into S. The Rb protein is a tumor-suppressor gene (more below).

The proteins induced by E2F include cyclin E, cyclin A, cdc25A (an activating protein phosphatase), and proteins required to bind at origins of replication to initiate DNA synthesis. The synthesis of cyclin E allows it to complex with cdk2, forming another active cyclin complex that retains activity into S phase (see Fig. 17.6). One of the major functions of the cyclin E1–cdk2 complex is hyperphosphorylation of the Rb protein, thereby keeping Rb in its inactive state. Cyclin A also complexes with cdk2, and it phosphorylates, and inactivates, the E2F family of transcription factors. This ensures that the signals are not present for extended periods of time. Thus, each phase of the cell cycle activates the next through cyclin synthesis. The cyclins are removed by regulated proteolysis.

Progression through the cell cycle is opposed by the CKIs (see Fig. 17.8). The CKIs regulating cyclin/cdk expression in the G₁ phase of the cell cycle fall into two categories: the Cip/Kip family and the INK4 family. The Cip/Kip family members (p21, p27, and p57) have a broad specificity and inhibit all cyclin–CDK complexes. The INK4 family, which consists of p15, p16, p17, and p19, are specific for the cyclin D–cdk4/6 family of complexes (inhibitors of cyclin-dependent kinase-4). The regulation of synthesis of different CKIs is complex, but some are induced by DNA damage to the cell and halt cell-cycle progression until the damage can be repaired. For example, the CKI p21 (a protein of 21,000 daltons) is a key member of this group that responds to specific signals to block cell proliferation. If the damage cannot be repaired, an apoptotic pathway is selected and the cell dies.

IV. Tumor-Suppressor Genes

Like the oncogenes, the tumor-suppressor genes encode molecules involved in the regulation of cell proliferation. Table 17.2 provides several examples. The normal function of tumor-suppressor proteins is generally to inhibit proliferation in response to certain signals such as DNA damage. The signal is removed when the cell is fully equipped to proliferate; the effect of the elimination of tumor-suppressor genes is to remove the brakes on cell growth. They affect cell-cycle regulation, signal transduction, transcription, and cell adhesion. The products of tumor-suppressor genes frequently modulate pathways that are activated by the products of proto-oncogenes.

TABLE 17.2 Examples of Tumor Suppressors

A. Tumor-Suppressor Genes That Regulate the Cell Cycle Directly

As discussed previously, the retinoblastoma gene (RB or RB1) product, Rb, functions in the transition from G₁ to S phase and regulates the activation of members of the E2F family of transcription factors (see Fig. 17.8). If an individual inherits a mutated copy of the RB1 allele, there is a 100% chance of that individual developing retinoblastoma, because of the high probability that the second allele of RB1 will gain a mutation (Fig. 17.9). This is considered familial retinoblastoma. Individuals who do not inherit mutations in RB1, but who develop retinoblastoma, are said to have sporadic retinoblastoma, and acquire two specific mutations, one in each RB1 allele of the retinoblast, during their lifetime.

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